Karina S. Cramer - US grants

DESCRIPTION (provided by applicant): The proposed research addresses potential molecular mechanisms underlying the formation of precise synaptic connections in the auditory brainstem. The applicant has previously shown that several members of the Eph family of receptor tyrosine kinases are expressed in avian auditory brainstem nuclei and along the midline during the formation of synaptic connections. These receptors mediate cell-cell interactions during axon outgrowth, and gradients of Eph receptors and their ligands, the ephrins, are required for the formation of precise topographic maps in the visual system. The Specific Aims are to test the roles of Eph receptor signaling in the development of auditory brainstem projections. Each group of experiments includes both descriptive studies of developmental expression and functional studies using experimental manipulations. Inovo microelectroporation will be used to misexpress Eph receptors and an in vitro labeling method will be used to evaluate resulting axonal projections. The generality of findings from chicks will be examined in developing mouse brainstem. Specific Aim 1 proposes experiments to investigate whether gradients of Eph receptors and ephrins are essential for the development of the tonotopic projection from VllIth nerve axons to the avian cochlear nucleus, n. magnocellularis. Specific Aim 2 extends these studies to mammals. The Eph receptor EphA4 is expressed nonuniformly in the developing mouse cochlear nuclei. The role of EphA4 in the establishment of a homologous projection from the VIIIth nerve to the cochlear nucleus will be evaluated in mutant mice that lack EphA4. Axonal projections from n. magnocellularis branch so that an ipsilateral branch synapses in the dorsal region of the target, n. laminaris, while the contralateral branch grows across the dorsal midline and contacts the ventral region of contralateral n. laminaris. Experiments outlined in Specific Aim 3 will evaluate the role of Eph receptors in the guidance of cochlear nucleus axons at the midline in the avian brainstem. These experiments will test whether Eph signaling is extensively used in the development of pathways in the auditory brainstem, and will address whether the roles for Eph receptors in establishing synaptic specificity are similar across modalities and across species.

The goal of this project is to develop an educational program in which undergraduate, graduate, and postdoctoral students learn the process of scientific discovery in the setting of active learning in biology coursework and hands-on experience in the principal investigator's laboratory. The research goal is to elucidate developmental mechanisms that give rise to complex sensory circuitry. The proposed research addresses important themes seen throughout the biology curriculum and will thus be integrated in the educational goal.

Sensory processing relies on neural circuitry that forms during embryonic and postnatal development in very precise patterns. A key question in biology is how these neural circuits form. In order to address this question, one must consider different levels of organization and how they are connected, a major theme in the PI's introductory biology course, From DNA to Organism. The PI uses the chick auditory system as a model in which to address this question. In particular, the circuit examined underlies the animal's ability to determine the locations of sounds. Neurons connect to each other through their axons, and axons can find their targets due to the integrated functions of proteins called axon guidance molecules. Of the many known axon guidance molecules, the Eph proteins are excellent candidates because previous studies by the PI demonstrate a role for one of these proteins, EphA4, in the accurate formation of connections in the auditory brainstem. Eph proteins are a large family of proteins with complex signaling mechanisms. The proposed research will investigate the role of two other candidate Eph proteins, EphB2 and ephrin-B1, and will examine the interactions between EphA4 and EphB2 during development. The PI has developed methods for introducing genes focally in the auditory brainstem and for tracing axonal connections between regions. These approaches allow for a careful and quantitative determination of which proteins are important for circuit formation, and where they act, and will provide a framework for investigating other classes of molecules.

The educational goal of the PI is a learning-centered approach to teaching biology. The proposed experiments will be performed by students, postdoctoral fellows, and technicians working collaboratively. This hypothesis-based inquiry is also reflected in the coursework taught by the PI. In From DNA to Organism, active learning approaches include group and individual activities to promote observation and information-gathering skills, and comprehension assessment in real-time. In the freshman seminar, discussions of developmental neuroscience are used to demonstrate how scientists obtain information and how skills for scientific inquiry are applied to careers in science. In addition, the PI will organize workshops for faculty and for postdoctoral fellows to provide a forum for developing approaches for active learning and using classroom technology effectively.

PROJECT SUMMARY Central auditory processing relies on precise networks of connections, beginning at the level of the auditory brainstem. The assembly of auditory circuitry during development requires multiple mechanisms to regulate axon growth, synaptogenesis, and pruning of axons and dendritic arbors. Neurodevelopmental disorders can lead to errors in these processes and can be associated with difficulties in auditory processing and communication. The overall goal of our research is to identify the cellular and molecular mechanisms that lead to precise formation of these auditory circuits. In particular, we are interested in identifying the functions of glial cells, non-neuronal cells that communicate with neurons and provide multiple functions throughout the developing and mature brain. We previously showed that glial cells influence synaptogenesis and dendritic maturation in the avian auditory brainstem. Here we expand on these findings to investigate the developmental roles of microglia in the mammalian auditory brainstem. Our studies will focus on three specific aims. First, we will determine the function of microglia in synaptic maturation and synaptic pruning in auditory pathways. We will use high resolution imaging approaches to investigate the roles of microglia and their signaling pathways in the formation of excitatory and inhibitory synapses in the auditory brainstem. Additionally, we will test whether microglia contribute to pruning of synapses in a specialized auditory pathway. Second, we will test determine the importance of microglia in auditory brainstem function. Using both genetic and pharmacological approaches to alter microglial signaling pathways or microglial numbers, we will determine the roles of microglia during development and maintenance of auditory brainstem responses. Third, we will investigate the role of microglia defining a critical period for lesion-induced synaptogenesis. We found that microglia peak in number in the medial nucleus of the trapezoid body at a time when this critical period closes. We will use genetic and pharmacological models to explore the contributions of microglia to developmental plasticity and to maturation of stable circuitry.